Preparation and application of specific antibody against coat protein (CP) of Chinese wheat mosaic virus (CWMV)

doi:10.3969/j.issn.1004-1524.20231119

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (9): 2042-2050.DOI: 10.3969/j.issn.1004-1524.20231119

• Plant Protection • Previous Articles Next Articles

Preparation and application of specific antibody against coat protein (CP) of Chinese wheat mosaic virus (CWMV)

SHEN Zhengrong¹^,²(), DAI Yuanxing², GUO Liuming², WANG Zhiyao¹^,², ZHANG Hengmu²^,^*()

1. College of Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
2. Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2023-09-18 Online:2024-09-25 Published:2024-09-30

Abstract

Abstract:

Chinese wheat mosaic virus (CWMV) is a soil-borne virus discovered and identified by Zhejiang Academy of Agricultural Sciences, whose genomic segment RNA2 encodes a major coat protein (CP) that plays an important role in virus infection. To detect its expression and analyze its biological function, the gene was amplified by RT-PCR from CWMV-infected wheat leaves. The recombinant expression vector was constructed with In-Fusion method and introduced into BL21(DE3) strain for the induction of CP expression. The recombinant CP protein was purified by nickel column affinity chromatography and injected into the rabbits for immunization. The antisera were purified by affinity chromatography and the polyclonal antibodies against CP were obtained. Dot-ELISA, ID-ELISA and Western blot assays showed that the purified antibodies were highly specific to CWMV with a titer of 1∶2.048×10⁷ and the sensitivity of 6.25×10^-2 ng. Dot-ELISA and Western blot assays showed that the antibody could be used for accurate diagnosis of CWMV, which laid the foundation for CWMV detection, CP quantitation and its functional research.

Key words: Chinese wheat mosaic virus, coat protein, recombinant protein, polyclonal antibody

CLC Number:

S435.12

SHEN Zhengrong, DAI Yuanxing, GUO Liuming, WANG Zhiyao, ZHANG Hengmu. Preparation and application of specific antibody against coat protein (CP) of Chinese wheat mosaic virus (CWMV)[J]. Acta Agriculturae Zhejiangensis, 2024, 36(9): 2042-2050.

Figures/Tables 7

Table 1 Primers used for PCR amplification

引物名称 Primer name	引物序列 Primer sequences (5'→3')	限制性内切酶 Restriction enzyme
CP-F	ATGGCCGTGAAATCTGG	—
CP-R	GCTCGAACCTTCCCACTTA	—
CP-F'	GCTGATATCGGATCCATGGCCGTGAAATCTGG	BamH I
CP-R'	TTGTCGACGGAGCTCGCTCGAACCTTCCCACTTA	Sac I
T7	TAATACGACTCACTATAGGG	—
T7-ter	GCTAGTTATTGCTCAGCGG	—

Fig.1 Construction and identification of recombinant vector pET-CP for expression in prokaryotic cells M, DNA Marker; Lane 1, Amplified fragment of CWMV CP gene; Lane 2, PCR products of empty pET-32a vector as negative control; Lanes 3-8, PCR products of recombinant pET-CP vectors; Lanes 9-11, Restriction enzyme digestion of PCR-positive pET-CP recombinant vectors with BamHⅠ/SacⅠ.

Fig.2 Assays for induced expression and affinity purification of recombinant CWMV CP protein A: M, Protein Marker; Lane 1, Uninduced host bacteria containing pET-32a; Lane 2, IPTG induced host bacteria containing pET-32a; Lane 3, Uninduced host bacteria containing pET-CP; Lanes 4-6, Host bacteria containing pET-CP induced by IPTG for 1-3 h. B: M, Protein Marker; Lane 1, Recombinant CP protein purified from host bacteria that containing pET-CP and induced by IPTG for 3 h.

Fig.3 Specificity analysis of antibody against CWMV CP A: M, Protein Marker; Lane 1, Induced host bacteria containing pET-32a; Lane 2, IPTG induced host bacteria containing pET-CP. B: M, Protein Marker; Lane 1, Healthy wheat sample; Lane 2, CWMV-infected wheat sample; Lane 3, BYDV-infected wheat sample; Lane 4, WYMV-infected wheat sample; Lane 5, ToMV-infected N. benthamiana sample; Lane 6, TuMV-infected N. benthamiana sample.

Fig.4 Titer of purified CP antibodies analyzed by indirect ELISA

Fig.5 Sensitivity of CP antibody detected by Dot-ELISA

Fig.6 Detection of the diseased wheat plants from fields by Dot-ELISA (A), Western blot (B) and RT-PCR(C) A: M, DNA Marker; Lane 1, Positive control; Lane 2, Negative control; Lanes 3-11, Samples of diseased wheat plants. B: M, Protein Marker; Lanes 1-9, Samples of diseased wheat plants. C: M, DNA Marker; Lane 1, Positive control; Lane 2, Negative control; Lanes 3-11, Samples of diseased wheat plants.

References 21

[1]	DIAO A, CHEN J, YE R, et al. Complete sequence and genome properties of Chinese wheat mosaic virus, a new furovirus from China[J]. The Journal of General Virology, 1999, 80 (5): 1141-1145.
[2]	YE R, ZHENG T, CHEN J P, et al. Characterization and partial sequence of a new furovirus of wheat in China[J]. Plant Pathology, 1999, 48(3): 379-387.
[3]	GUO L M, HE J, LI J, et al. Chinese wheat mosaic virus: a long-term threat to wheat in China[J]. Journal of Integrative Agriculture, 2019, 18(4): 821-829.
[4]	孙炳剑, 羊健, 孙丽英, 等. 禾谷多黏菌传小麦病毒病的分布及变化动态[J]. 麦类作物学报, 2011, 31(5): 969-973.
	SUN B J, YANG J, SUN L Y, et al. Distribution and dynamics of Polymyxa graminis transmitted wheat viruses in China[J]. Journal of Triticeae Crops, 2011, 31(5): 969-973. (in Chinese with English abstract)
[5]	OHKI T. First report of barley virus G infecting wheat (Triticum aestivum) in Japan[J]. Plant Disease, 2023, 107(8): 2569.
[6]	CHEN J P. Occurrence of fungally transmitted wheat mosaic viruses in China[J]. Annals of Applied Biology, 1993, 123(1): 55-61.
[7]	ESTES A P, BRAKKE M K. Correlation of Polymyxa graminis with transmission of soil-borne wheat mosaic virus[J]. Virology, 1966, 28(4): 772-774.
[8]	YANG J, CHEN J, CHEN H, et al. Sequence of a second isolate of Chinese wheat mosaic furovirus[J]. Journal of Phytopathology, 2001, 149(3/4): 135-140.
[9]	XU L, CHEN J P, YE R, et al. Cloning and expression of the Chinese wheat mosaic virus RNA2 coat protein readthrough and 19 ku cysteinerich domains and localization of these proteins[J]. Chinese Science Bulletin, 2002, 47(12): 1019-1023.
[10]	YANG J, ZHANG F, XIE L, et al. Functional identification of two minor capsid proteins from Chinese wheat mosaic virus using its infectious full-length cDNA clones[J]. The Journal of General Virology, 2016, 97(9): 2441-2450.
[11]	ZHU B G, CAI G F, HALL E O, et al. In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations[J]. BioTechniques, 2007, 43(3): 354-359. PMID
[12]	ABOU-ELHAKAM H M, FARID A A, MAHANA N A, et al. Dot-ELISA as a field test for hydatid diagnosis[J]. Journal of the Egyptian Society of Parasitology, 2016, 46(2): 441-452.
[13]	IVANOV K I, MÄKINEN K. Coat proteins, host factors and plant viral replication[J]. Current Opinion in Virology, 2012, 2(6): 712-718. DOI PMID
[14]	杨锦, 靳鹏, 刘芃, 等. 表达中国小麦花叶病毒(CWMV)外壳蛋白基因增强烟草对CWMV的抗病性[J]. 浙江农林大学学报, 2020, 37(2): 291-295.
	YANG J, JIN P, LIU P, et al. Transgenetic expression coat protein of Chinese wheat mosaic virus(CWMV) enhances resistance of Nicotiana benthamiana to CWMV[J]. Journal of Zhejiang A & F University, 2020, 37(2): 291-295. (in Chinese with English abstract)
[15]	NIU E B, YE C Z, ZHAO W Y, et al. Coat protein of Chinese wheat mosaic virus upregulates and interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenase, a negative regulator of plant autophagy, to promote virus infection[J]. Journal of Integrative Plant Biology, 2022, 64(8): 1631-1645. DOI
[16]	娄虎, 徐熔, 王海竹, 等. 植物病毒病检测及防治的研究进展[J]. 江苏农业科学, 2017, 45(24): 25-31.
	LOU H, XU R, WANG H Z, et al. Research progress on detection and control of plant virus diseases[J]. Jiangsu Agricultural Sciences, 2017, 45(24): 25-31. (in Chinese)
[17]	徐磊, 叶荣, 赵秀云, 等. 中国小麦花叶病毒RNA2外壳蛋白通读区及19ku富半胱氨酸基因的克隆、表达及其编码蛋白定位[J]. 科学通报, 2002, 47(6): 439-443.
	XU L, YE R, ZHAO X Y, et al. Cloning, expression and protein localization of China wheat mosaic virus RNA2 coat protein read-through region and 19ku cysteine-rich gene[J]. Chinese Science Bulletin, 2002, 47(6): 439-443. (in Chinese)
[18]	任春梅, 程兆榜, 朱慧, 等. 3种麦类土传花叶病毒的多抗制备及检测应用[J]. 江苏农业科学, 2014, 42(11): 143-146.
	REN C M, CHENG Z B, ZHU H, et al. Preparation and detection of polyclonal antibodies against three wheat soil-borne mosaic viruses[J]. Jiangsu Agricultural Sciences, 2014, 42(11): 143-146. (in Chinese)
[19]	孔凡惠, 脱建波, 魏娇, 等. 中国小麦花叶病毒CP和CRP蛋白的原核表达、抗血清制备及RNA2侵染性克隆构建[J]. 山东农业科学, 2015, 47(8): 6-10.
	KONG F H, TUO J B, WEI J, et al. Prokaryotic expression and antiserum production of Chinese wheat mosaic virus(CWMV) CP and CRP and construction of their RNA2 infection clones[J]. Shandong Agricultural Sciences, 2015, 47(8): 6-10. (in Chinese with English abstract)
[20]	刘欢, 李娜, 周雪平, 等. 中国小麦花叶病毒(CWMV)单克隆抗体制备及其检测应用[J]. 农业生物技术学报, 2015, 23(6): 711-719.
	LIU H, LI N, ZHOU X P, et al. Development of monoclonal antibodies against Chinese wheat mosaic virus(CWMV) and their application[J]. Journal of Agricultural Biotechnology, 2015, 23(6): 711-719. (in Chinese with English abstract)
[21]	PAN S H, MALCOLM B A. Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3)[J]. BioTechniques, 2000, 29(6): 1234-1238. PMID

Preparation and application of specific antibody against coat protein (CP) of Chinese wheat mosaic virus (CWMV)

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 7

References 21

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	YAN Jingying, NI Liang, SHEN Xingyu, LI Yu. Effect of heat treatment on the degradation of recombinant protein and recombinant DNA in transgenic straws [J]. Acta Agriculturae Zhejiangensis, 2024, 36(9): 2079-2088.
[2]	LI Tian’en, ZHOU Sihan, SUN Hongchao, FU Yuan, SHI Tuanyuan, YAN Wenchao. Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens [J]. Acta Agriculturae Zhejiangensis, 2024, 36(3): 503-514.
[3]	TANG Guoliang, ZHANG Yubao, WANG Ruoyu, WANG Yajun, ZHAO Xia, SU Xuesi, JIN Weijie. Prokaryotic expression of Konjac mosaic virus capsid protein and preparation of polyclonal antibody [J]. Acta Agriculturae Zhejiangensis, 2022, 34(11): 2471-2481.
[4]	LIU Shuya, HE Wenlu, TAO Yu, HUANG Shuanghui, REN Yongqiang, GENG Yi, HUANG Xiaoli, CHEN Defang, OUYANG Ping. Preparation and immunoprotection of ORF57 subunit vaccine of Cyprinid herpesvirus 3 [J]. Acta Agriculturae Zhejiangensis, 2022, 34(10): 2138-2148.
[5]	ZHU Yinchu, WANG Hongyu, YUN Tao, HUA Jionggang, YE Weicheng, NI Zheng, CHEN Liu, ZHANG Cun. Isolation and identification of goose astrovirus in Zhejiang Province, China, and preparation of polyclone antibodies against capside protein [J]. Acta Agriculturae Zhejiangensis, 2022, 34(10): 2149-2159.
[6]	SU Xuesi, ZHANG Yubao, WANG Ruoyu, WANG Yajun, TANG Guoliang, JIN Weijie. Prokaryotic expression of Plantago asiatica mosaic virus capsid protein and preparation of its polyclonal antibody [J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 104-111.
[7]	LIU Jinyu, HUANG Ying. Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe [J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 34-42.
[8]	PAN Chuanyan, LIN Yong, FENG Pengfei, ZHANG Yongde, LUO Honglin. Prokaryotic expression and polyclonal antibody preparation of Hsc70 in Nile tilapia [J]. , 2019, 31(8): 1272-1279.
[9]	YAN Han, YANG Ruixiu, GAI Xiaotong, LI Gang, ZHAO Zhiguo, GAO Zenggui. Establishment of ELISA for Fusarium oxysporum f. sp. melonis [J]. , 2018, 30(12): 2081-2086.
[10]	ZHANG Chengling, SUN Houjun, YANG Dongjing, XU Zhen, ZHAO Yongqiang, XIE Yiping. Molecular variation and genetic diversity analysis of coat protein gene in Sweetpoviruses in China [J]. , 2017, 29(4): 611-617.
[11]	BI Zhuangli1，2， ZHU Yingqi1，2， CHEN Zongyan2， LI Chuanfeng2， MENG Chunchun2， WANG Guijun1， LIU Guangqing2，*. Prokaryotic expression and polyclonal antibody preparation of σC protein of novel duck reovirus [J]. , 2014, 26(4): 882-.
[12]	WANG Jing\\|xiu;GAO Zhang\\|zhao;JIANG Yong\\|hou;;WU Hai\\|gang;RAO Pin\\|bin;QUAN Yan\\|ping;XU Hui. Prokaryotic expression of the PCV2 ORF4 gene and preparation of polyclonal antibodies against ORF4 WANG Jing\\|xiu1， GAO Zhang\\|zhao1， JIANG Yong\\|hou1,， WU Hai\\|gang1， RAO Pin\\|bin1， QUAN Yan\\|ping1， XU Hui2 [J]. , 2014, 26(1): 0-39.
[13]	DU Zhi-qiang. Expression， purification and polyclonal antibody preparation of recombinant Lv-PEN-3 protein [J]. , 2013, 25(5): 0-940.
[14]	DU Zhi-qiang;CHENG Jia;WANG Jian-ying*. Purification and antibody preparation of rBLS recombinant protein from swine Brucella [J]. , 2013, 25(3): 0-482.
[15]	ZHAO Li-na;CUI Yan-shun;REN Jian-ting;LI Jian-liang;HUANG Bing;LI Yu-feng;MA Xiu-li;*. Cloning and expression of VP1 gene of Duck hepatitis virus genotype C and preparation of its polyclonal antibody [J]. , 2011, 23(2): 0-262.